Optical analysis system and approach therefor

ABSTRACT

Imaging, testing and/or analysis of subjects are facilitated with a capillary-access approach. According to an example embodiment, a capillary is implanted into a specimen and adapted to accept an optical probe to facilitate optical access into the specimen. In some applications, the capillary is implanted for use over time, with one or more different probes being inserted into the capillary at different times, while the capillary is implanted. Certain applications involve capillary implantation over weeks, months or longer. Other applications are directed to the passage of fluid to and/or from a sample via the capillary. Still other applications are directed to the passage of electrical information between the sample and an external arrangement, via an implanted capillary.

RELATED PATENT DOCUMENTS

This patent document is a continuation under 35 U.S.C. § 120 of U.S.patent application Ser. No. 15/967,211 filed on Apr. 30, 2018, which isa continuation of U.S. patent application Ser. No. 15/830,894 filed onDec. 4, 2017 (abandoned), which is a continuation of U.S. patentapplication Ser. No. 14/876,620 filed on Oct. 6, 2015 (U.S. Pat. No.9,389,361), which is a continuation of U.S. patent application Ser. No.13/540,897 filed on Jul. 3, 2012 (U.S. Pat. No. 9,161,694), which is acontinuation of U.S. patent application Ser. No. 11/334,769 filed onJan. 18, 2006 (U.S. Pat. No. 8,346,346), and which claims the benefitunder 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No.60/646,858, entitled “Optical Analysis Systems and Approaches” and filedon Jan. 24, 2005.

FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under contract DA017895awarded by the National Institutes of Health. The U.S. Government hascertain rights in this invention.

FIELD OF THE INVENTION

The present invention relates generally to optical analysis, and moreparticularly to analysis approaches involving the optical analysis ofsubjects accessed via a capillary.

BACKGROUND

A variety of approaches to optical imaging and analysis have been usedfor many different applications. For example, the endoscope is a usefultool for a variety of applications such as biological research, medicaldiagnostics, and for image guidance in surgical procedures. Conventionalendoscopes utilize a white light source to illuminate a sample andreflected light to visualize the same sample. Such conventionalendoscopes are typically limited, however, to visualizing the surface ofa sample or to surface inspection within a hollow tissue cavity.

Certain types of optical analysis approaches are discussed in connectionwith the following patent documents: U.S. Pat. Nos. 6,485,413 and6,423,956, and U.S. Patent Application Publication Number US2003/0142934.

Many applications for which optical analysis would be beneficial aresubject to a variety of limitations to such analysis. For example, spaceconstraints in many applications limit the use of certain tools that arenot generally scaleable in a manner that would facilitate such tools'use for these applications. In addition, while certain tools have beenuseful in applications characterized by small space constraints, thesetools are often limited in their ability to achieve desirable results,or by their ability for use with certain samples such as biologicalsamples that may include live beings. Furthermore, many optical analysisapproaches are limited to the analysis of linear optics.

Some applications benefit from subcutaneous analysis, in particular witha specimen. However, invasive analysis of a specimen can be challenging,particularly when the analysis is to be made over time. For example,when a subcutaneous area of a living specimen is to be accessed multipletimes, processes used to facilitate the access must be repeated. Inaddition, each time a specimen is accessed subcutaneously, infection andother medical complications can arise.

The above and other issues have presented challenges to optical analysisapproaches and, in particular, to optical imaging in applicationsexhibiting relatively small space such as for endoscopic and microscopicapplications.

SUMMARY OF THE INVENTION

The present invention is directed to overcoming the above-mentionedchallenges and others related to the types of devices and applicationsdiscussed above and in other applications. These and other aspects ofthe present invention are exemplified in a number of illustratedimplementations and applications, some of which are shown in the figuresand characterized in the claims section that follows.

According to an example embodiment, a capillary arrangement is adaptedfor insertion and coupling to a specimen for optical analysis. Thecapillary includes a lens-type structure near a lower end thereof, whichis inserted into the specimen. Walls of the capillary facilitate anopening into the specimen and to a depth at the lower end, with anopening in an upper portion of the capillary facilitating access intothe capillary and into a region of the specimen optically accessible viathe lower end of the capillary.

According to another example embodiment, a glass capillary having anopening at both ends thereof is attached to a polished coverslip that isaffixed to one end of the glass capillary. The end of the glasscapillary with the polished coverslip affixed thereto is inserted into aspecimen to facilitate optical access into the specimen via thecoverslip. The capillary and coverslip accept one or more of a varietyof probes inserted thereto and that access the specimen via thecoverslip. The arrangement of the capillary and coverslip thus allowaccess into the specimen (e.g., subcutaneous access) without necessarilyexposing tissue in the specimen to a probe or other materials orconditions in or around the capillary.

According to another example embodiment of the present invention, anoptical system facilitates accessing to and analysis of an internalportion of a sample. The system includes an optical access arrangementhaving an embedded end in the sample and an exposed end extending atleast to an external surface of the sample. Sidewalls extend from theembedded end to the exposed end, where the sidewalls and the embeddedend separate an internal portion of the sample from an externalenvironment located at the exposed end and within the sidewalls. Thatis, an area within the sidewalls and bounded at the embedded end isexposed to air or other environmental conditions at the exposed end. Theoptical access arrangement is further adapted to accept an optical probewithin its sidewalls and extending to the embedded end, such as with aprobe inserted from a location at the exposed end and into the sample;the sidewalls and embedded end thus separate the probe and the externalenvironment from an internal portion of the sample. A lens-typearrangement is at the embedded end of the optical access arrangement andfacilitates the passage of light into the sample. A light directordirects light, via the lens-type arrangement, to a target location inthe sample and further directs light from the target location to a lightdetector. Such a light director may, for example, include an opticalfiber, one or mirrors, or other devices or components that facilitatethe passage of light. A light detector receives light from the sample,via the light director, and presents a signal characterizing thedetected light.

According to another example embodiment of the present invention, anoptical capillary arrangement is for implantation into subcutaneoustissue of a being. The arrangement includes a lower distal end separatedfrom an upper distal end by sidewalls extending therebetween, and isadapted to accept an optical probe extending to a subcutaneous depthinto the being. The lower distal end and sidewalls separate subcutaneoustissue of the live being from an external environment within thesidewalls upon implantation. The sidewalls include an exchange passageadapted to exchange at least one of fluid and electrical signals betweena target region in the subcutaneous tissue and an environment at theupper distal end. In this context, the exchange passage may include oneor more of an electrically conducting material and/or a fluid-passingtube or membrane.

According to another example embodiment of the present invention, amethod for analyzing subcutaneous tissue of a live being involvesinserting a lower distal end of a capillary into the subcutaneoustissue. The capillary has sidewalls extending from the lower distal endto an upper distal end, wherein the upper distal end remains exposedover a surface of the sample after insertion of the lower distal end.The subcutaneous tissue is separated from an environment in thecapillary by the lower distal end and the sidewalls. An optical probe isinserted into the capillary and the subcutaneous tissue is imaged. Theoptical probe is removed and the live being is allowed to move freely.At a time after removing the optical probe and allowing the live beingto move freely, an optical probe is re-inserted into the capillary andthe subcutaneous tissue is re-imaged.

The above summary is not intended to describe each illustratedembodiment or every implementation of the present invention. The figuresand detailed description that follow more particularly exemplify theseembodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be more completely understood in consideration of thedetailed description of various embodiments of the invention thatfollows in connection with the accompanying drawings in which:

FIG. 1 shows a system for optical imaging, according to an exampleembodiment of the present invention;

FIG. 2 shows a cross-sectional view of an arrangement for opticalimaging, according to another example embodiment of the presentinvention;

FIG. 3 shows a cross-sectional view of a capillary-type arrangement foranalyzing a sample, according to another example embodiment of thepresent invention;

FIG. 4 shows a system for optical imaging with fluid and electricalcapabilities, according to another example embodiment of the presentinvention; and

FIG. 5 shows an end view of a capillary arrangement such as thatimplemented in connection with FIG. 4, according to another exampleembodiment of the present invention.

While the invention is amenable to various modifications and alternativeforms, specifics thereof have been shown by way of example in thedrawings and will be described in detail. It should be understood,however, that the intention is not to limit the invention to theparticular embodiments described. On the contrary, the intention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention.

DETAILED DESCRIPTION

The present invention is believed to be applicable to a variety ofdifferent types of devices and approaches, and the invention has beenfound to be particularly suited for approaches to optical analysisinvolving the imaging of beings, and is applicable to in-vivo analysis.While the present invention is not necessarily limited to suchapplications, various aspects of the invention may be appreciatedthrough a discussion of various examples using this context.

According to an example embodiment of the present invention, an opticalanalysis arrangement includes an implantable capillary-type device thatfacilitates acute (single) and/or iterative access to internal areas(e.g., tissue) of a specimen via an externally-applied probe, such as anoptical probe inserted into the capillary-type device from outside thespecimen. The capillary-type device is adapted to couple to a specimen,such as a living being, and to remain in an implanted state. A portionof the capillary implanted in the specimen and adjacent a target areafor testing is adapted to pass light between an external probe-typearrangement and the target area in the specimen.

In another example embodiment of the present invention, an opticalanalysis capillary-type approach facilitates the optical analysis of asample such as a biological specimen or a living being, for single oriterative analysis applications. A capillary is configured for acceptingan optical probe, such as an endoscopic probe via an open end of thecapillary, with a closed end of the capillary facilitating the passageof light between the specimen and the probe. A portion of the capillaryincluding the closed end thereof is implanted into a sample, with theopen end accessible for insertion of an optical probe.

In some instances, an optical probe implemented with one or more of thecapillary-type approaches described herein is a microendoscope probe,such as those based on GRIN lenses. Various example embodiments aredirected to the user of GRIN lens applications similar to thosedescribed in the following U.S. patent publications, each of which listsMark Schnitzer as an inventor thereof: No. 20040260148 entitled“Multi-photon endoscopic imaging system”; No. 20040143190 entitled“Mapping neural and muscular electrical activity”; No. 20030118305entitled “Grin fiber lenses”; No. 20030117715 entitled “Graded-indexlens microscopes”; No. 20030031410 entitled “Multi-photon endoscopy”;No. 20020146202 entitled “GRIN fiber lenses”; and No. 20020141714entitled “Grin-fiber lens based optical endoscopes”; all of which arefully incorporated herein by reference.

In other particular instances, an optical probe implemented with one ormore of the capillary-type approaches described herein includes opticalfibers, and in some applications, a bundle of optical fibers. Variousexample embodiments are directed to the use of optical fibers such asthose described in the following U.S. Patent Publications: No.20050157981 entitled “Miniaturized focusing optical head in particularfor endoscope” (to Berier et al.), No. 20050207668 entitled “Method forprocessing an image acquired through a guide consisting of a pluralityof optical fibers” (to Perchant, et al.), No. 20050242298 entitled“Method and equipment for fiber optic high-resolution, in particularconfocal, fluorescence imaging” (to Genet, et al.) and No. 20030103262entitled “Multimodal miniature microscope” (to Richards-Kortum, et al.);and as those described in the following U.S. Pat. No. 6,766,184(Utzinger, et al.) entitled “Methods and apparatus for diagnosticmultispectral digital imaging,” U.S. Pat. No. 6,639,674 (Sokolov, etal.) entitled “Methods and apparatus for polarized reflectancespectroscopy,” U.S. Pat. No. 6,571,118 (Utzinger et al.) entitled“Combined fluorescence and reflectance spectroscopy,” and U.S. Pat. No.5,929,985 (Sandison, et al.) entitled “Multispectral imaging probe,” allof which are fully incorporated herein by reference.

In a more particular example embodiment, a capillary as discussed in thepreceding examples is further adapted to facilitate the micro-fluidicdelivery and sampling to and/or from a specimen. Supplied fluids mayinclude, for example, fluids that facilitate the detection of opticalcharacteristics of a target location in the specimen, or fluids thatinclude drugs and/or other substances that affect the specimen.Micro-fluidic transfer from a specimen may involve, for example, onlinerecovery of fluid specimens for off-line genetic, biochemical, chemical,genomic, proteomic, or cytometric analyses. In some embodiments, fluidlines are permanently implanted in a specimen along with the capillary.In other embodiments, the fluid lines are inserted into the specimen(and reinserted for subsequent analysis) via specific routes in thecapillary.

In another example embodiment, a capillary as discussed in the precedingexamples is further adapted to pass a stimulus into a specimen tofacilitate the detection of characteristics of the specimen and, in someinstances, the characteristics of a particular target area in thespecimen. In one implementation, the stimulus is an electrical stimulus,passed from the capillary to a target area. One or more electrodes at animplanted portion of the capillary facilitate the passage of theelectrical stimulus into the specimen. In another implementation,electrodes at an implanted portion of the capillary are used tofacilitate in vivo recordings of nervous, cardiac, or muscular activityat the imaging field. In another implementation, a chemical stimulus,such as that delivered via a fluid as discussed above, is applied to thetarget area via the capillary. In some embodiments, electrodes arepermanently implanted in the specimen along with the capillary, and inother embodiments, the electrodes are inserted into the specimen (andreinserted for subsequent analysis) via specific routes in thecapillary.

In other example embodiments, the capillary includes optical devicessuch as lenses, filters, polarizers or other devices that facilitate thepassage of light between a probe and a sample. In certain exampleembodiments, the capillary is implemented with a cover slip, or sheath,that facilitates implantation into a specimen. In another exampleembodiment, at least some of the capillary is coated or otherwiseconfigured with material that inhibits certain adverse characteristicsof invasive implantation into live beings.

In another example embodiment of the present invention, an approach tooptical analysis involves the use of a micro-mirror arrangement,including one or more mirrors, integrated with a light directingcapillary-type arrangement to facilitate optical stimulation andresponse detection with a sample. The micro-mirror arrangement is usedto direct light to a sample, with light from the sample being collectedand used for analyzing the sample (i.e., detecting a response, orcondition, of the sample). The micro-mirror arrangement is selectivelyimplemented in the capillary-type arrangement, facilitating near-targetplacement of the micro-mirror arrangement while also enabling access tothe micro-mirror arrangement from an external position, withoutnecessarily removing the capillary-type arrangement or otherwisedisturbing the sample.

In some applications, the micro-mirror arrangement includes a MEMS(micro-electro-mechanical systems) mirror, and the optical analysisapproach therewith involves the optical stimulation of a sample togenerate nonlinear optical responses. Certain approaches are thusimplemented to facilitate the detection of a nonlinear response. Forexample, certain stimulation approaches known to generate nonlinearoptical responses are used in connection with a sample and the scanningof the sample with the MEMS mirror. Detected nonlinear optical responsesto the scanning are made available for use in analyzing the sample.

In certain implementations, arrangements with which the MEMS mirror isused are configured for directing response light of a wavelengthdifferent than that of stimulation light, to facilitate the detectionand analysis thereof. In one application, a light detector and/or alight director (something that directs light) is implemented to analyzewavelength-related characteristics of a response of the sample via animplanted capillary-type arrangement. For instance, where light of aparticular wavelength is directed to the sample via a MEMS mirror, lightof a different wavelength is collected from the sample as a response,and analyzed accordingly. Certain characteristics of a light collector(or detector) are selectively tailored to facilitate the detection oflight at such a different wavelength. Some applications involve the useof a wavelength-dependent dichroic element, such as a beam splitter,that directs light of a particular wavelength (e.g., the wavelength oflight related to a response of the sample) to a detector.

In another example embodiment of the present invention, a light directoris adapted for analyzing a sample using a movable micro-mirror and adichroic device to selectively pass stimulation and response light. Themicro-mirror is moved using an actuator, such as a pivoting ortranslating actuator, for controllably moving the mirror to direct lightfrom a light source, such as a laser or incandescent source, todifferent target locations on the sample. The dichroic device includesone or more wavelength-selective components, such as a beam splittingprism, a dichroic mirror or a curved dichroic beam splitter. Such adichroic device is selectively placed in the capillary-type arrangement,where appropriate, for selective wavelength-dependent direction of lightinto and/or from a sample. In some applications, the dichroic device isused to separate wavelengths that are subsequently used to generate amulti-channel image.

According to another example embodiment of the present invention, anoptical system is adapted for analyzing a sample. The system includes alight source, a light director, an implantable capillary-typearrangement, a dichroic device and a light detector. The light directorincludes a micro-mirror, an actuator and a dichroic device, and isselectively located in the capillary-type arrangement. The actuator(e.g., a MEMS motor) is adapted to controllably move the mirror, such asby pivoting the mirror about one or more axes or moving the mirrorlaterally and/or vertically relative to the sample. The mirror iscontrollably moved to direct light from the light source to differenttarget locations on the sample. In some applications, the actuator isadapted to move the micro-mirror in a manner that causes light to scanacross a target area in the sample (i.e., via an embedded lens at alower end of a capillary-type arrangement).

The dichroic device is adapted to pass light from the light source tothe sample and to direct response light from the sample to a lightdetector (e.g., using wavelength-dependent characteristics of thedichroic device to selectively direct the response light to the lightdetector). The dichroic device may be implemented using one or more of avariety of approaches, such as those involving a beam splitter or otherwavelength-selective component as discussed herein.

The light detector is configured and arranged to receive light from thesample, via the capillary-type arrangement and the dichroic device, andto present a signal characterizing the detected light. The lightdetector is implemented using one or more of a variety of devices, suchas a photo detector, light collector, photo multiplier tube, imagingelectronics, lenses and others as discussed, e.g., in connection withother embodiments and implementations herein.

Turning now to the Figures, FIG. 1 shows an optical analysis system 100,according to another example embodiment of the present invention. Theoptical analysis system 100 includes a light source 110 and a capillary120 arranged to facilitate the selective direction of light from thelight source to a target location or locations on a sample 105 (e.g., aspecimen). In some applications, the light source 110 is a laser. Avariety of lasers can be used with the light source 110, with a fewexamples including an Argon ion laser, a YAG laser, a laser diode, aTitanium sapphire laser, a superluminescent diode and aChromium-fosterite laser. Target locations may include, for example,locations of interest for a particular type of analysis or specificlocations relating to implanted devices, such as probes, that facilitatethe detection of a response from the sample.

Stimulation light from the light source 110 is directed to the capillary120 via a beam splitter 115 (or similar device). Response light from thesample 105 (e.g., reflected and/or refracted light) is returned to asensing arrangement 130 via the beam splitter 115. The detector 130 may,for example, include a photo-multiplier tube (PMT), a CCD camera or anyarrangement that can process light to generate an output that can beused to characterize, and/or to produce an image from, the light fromthe sample. In some implementations, a processor 132 such as a computeror other device is used to characterize the light and/or to reconstructa digital image in such a manner.

In some applications, mirrors are implemented in connection with FIG. 1to direct light to the sample 105 via a lower end of the capillary 120,and include a variety of structures and implement a variety ofapproaches, depending upon the particular application. In one example, aMEMS mirror is implemented in connection with the capillary 120, tofacilitate light direction, scanning and/or other light-relatedapplication functions.

A light conduit 150, such as a fiber optic conduit, is optionallyimplemented to facilitate the transport of light from the beam splitter115 into the capillary 120 and to the target location. In someapplications, the light conduit 150 is a single conduit and, in otherapplications, the light conduit 150 includes two or more light conduitsthat are selectively implemented, e.g., for passing light to and/or fromthe sample 105. Similar light conduits 152 and 154 are selectivelyimplemented with the transport of light respectively from the lightsource 110 to the beam splitter 115, and from the beam splitter 115 tothe light sensing arrangement 130.

In some particular instances, the light conduit 150 is a microendoscopeprobe, such as those based on GRIN lenses as described in the U.S.Patent documents listing Mark Schnitzer as an inventor and cited above.In other particular instances, the light conduit 150 is an optical probehaving a bundle of optical fibers, such as those described in the U.S.patents and patent Publications referenced above in connection withfiber applications.

FIG. 2 shows a cross-sectional view of a capillary arrangement 200,according to another example embodiment of the present invention. Thecapillary arrangement 200 may, for example, be implemented in connectionwith the capillary 120 in FIG. 1. The capillary arrangement 200 includesa capillary 210, or guiding tube, having a lower closed end 212 and anupper open end 214, with a sidewall 216. Optionally, one or more opticalelements 220 (e.g., a lens-type arrangement that focuses or simplypasses light, numbered 1-n by way of example) are included near theclosed end 212 of the capillary 210. Here, the capillary 210 is shown ina generally cylindrical shape; however, the shape and correspondingarrangement of the side wall 216 can be implemented with a variety ofshapes for different applications.

In some applications, the optical elements 220 are implemented with asingle glass optical element, which is an optical flat of about uniformthickness. The thickness of this single glass optical element isselected to meet particular applications using considerations such asthose related to the desire to prevent fracture or damage to the flatand/or to the desire to reduce the optical distance between the analyzedtissue and an optical probe inserted into the capillary. In oneparticular application, a glass optical element having a thickness ofbetween about 50 and 500 microns is used to facilitate the aforesaidconsiderations.

The capillary 210 is adapted for implantation in a sample, such as atissue sample and/or a live being, with the upper open end 214 extendingto an accessible location (i.e., outside and/or at a surface of thesample or live being). After implantation, an optical probe 230 such asan endoscope can be inserted into the exposed upper open end 214 of thecapillary 210 for passing light via the closed end 212 to the sample inwhich the capillary 210 is implanted.

The closed end 212 of the capillary 210 generally facilitates thepassage of light, and may include one or more of a polished end, gluedend, plastic end, ceramic end, glass end and others. In someapplications, the closed end 212 includes one or more passageways thatare selectively implemented for the passage of fluids to and/or from aspecimen in which the capillary 210 is implanted. In some embodiments,the fluid lines are permanently implanted in a specimen along with thecapillary, and in other embodiments, the fluid lines are inserted intothe specimen (and reinserted as appropriate for subsequent analysis) viaspecific routes in the capillary.

In one application, the capillary 210 has a generally cylindrical shape,with inner and outer diameters (as defined by the sidewall 216)respectively about 1.4 mm and 1.6 mm. A generally round light-passingpiece is glued at the lower end 212 of the capillary 210 and thearrangement is cured. A fiber grinder is used to polish the capillary,in particular the light-passing piece and the glued lower end 212 of thecapillary 210.

In some implementations, the capillary is adapted for implantation intoa live being to facilitate optical analysis over the course of time,with the capillary left implanted in the being during times in-betweeninstances of optical analysis. For example, where tissue in a live beingis to be studied, the capillary is implanted in the being such that theclosed end thereof facilitates the passage of light to and from targetareas of the tissue. With this approach, the upper open end 214 of thecapillary 210 is available for the insertion of a probe thereto,facilitating access into the live being with the probe from a relativeexternal position. In some instances, the open end 214 of the capillary210 may be accessible via outer tissue of the being, such as via theskin or skull tissue of the being. In other instances, the open end 214of the capillary 210 is inside of the live being, but accessible by aprobe (e.g., where the capillary is implanted in a region accessible viaa blood vessel or via non-invasive or invasive type approaches).

Once implanted, the capillary 210 is used to selectively opticallyaccess tissue near the capillary over the course of hours, days, weeks,months or more. In this regard, the study of a live being over time isfacilitated, such as for the reaction of the live being to a particulartreatment. For example, drugs can be administered to the live beingunder analysis and the response thereto is readily detected, over time,via the capillary.

In certain applications, additional functional components such as fluidlines, electrodes and others are implemented with the capillary 210.These components may be implemented, for example, to stimulate orotherwise affect the tissue adjacent to the capillary 210, such as byelectrically stimulating the tissue or by supplying drugs, dyes or otherfluids to the tissue. These components can also be used to monitor thetissue, such as by facilitating electrophysiological recordings orfluidic sampling of specimens to be analyzed using genetic, biochemical,cytometric or other approaches.

The one or more optical elements 220 include one or more of a variety oflight-passing arrangements. In one implementation, the one or moreoptical elements 220 include gradient refractive index (GRIN) opticssuch as a GRIN lens or GRIN lens array that facilitates the collimationof light. In another application, the optical elements 220 include avariable focal length lens, adaptable for focusing upon subjects atdifferent focal depths. In other applications, the optical elementsinclude a liquid lens.

FIG. 3 is a capillary-type arrangement 300 that selectively facilitateselectrical and/or fluidic interaction with a sample, in connection withanother example embodiment of the present invention. The electricaland/or fluidic interaction may, for example, involve providing stimulusto the sample, detecting an electrical characteristic of the sample orretrieving fluid from the sample. The arrangement 300 includes acapillary 310, similar to the capillary 210 shown in FIG. 2, with alower end 312 adapted for implanting into a sample and the passage oflight into and out of the sample. As with the capillary 210, thecapillary 310 facilitates the use of a light director 330 such as afiber optic device for delivery of light into the sample and/or passageof light from the sample to an external light detector. In this regard,the lower end 312 includes a lens or other arrangement that facilitatesthe passage of light and may, for example, include multiple lenses orother arrangements that enhance or otherwise effect the passage oflight. In certain applications involving one or more lenses, a MEMSactuator 313 (shown by dashed lines) is coupled to a lens at the lowerend 312 and adapted to move the lens as shown by directional arrows 315to focus upon selected targets.

In another example embodiment, the arrangement 300 further includes afluidic exchange 340 that facilitates the introduction and/or withdrawalof fluids to/from a sample in which the lower end 312 is embedded.Drugs, stimulating fluids, cleaning fluids or other fluids thatfacilitate a particular type of analysis of a specimen can be passedinto the sample. This fluid supply may facilitate an optical responsefrom the sample, which can be detected via the light director 330. Incertain applications, two or more fluid exchange arrangement are thusimplemented (e.g., with one for fluid supply and another for fluidwithdrawal).

In some applications, the fluid exchange 340 draws fluid from a sampleto enhance optical analysis approaches, such as by removing particles orother objects/fluid that may hinder optical analysis via the capillary310. This fluid may include cells imaged or at a target region to whichthe arrangement 300 is directed, and in some applications includes othercells from adjacent areas. Moreover, this fluid withdrawal isfacilitated over time, during optical analysis, without necessarilyre-accessing the sample (i.e., access to the arrangement 300, implantedfor a period of time, is facilitated via the capillary 310).

In various embodiments, off-line analysis approaches involve fluidsampling to facilitate one or more of genetic testing using gene chips,biochemical analyses such as microdialysis, and cytometric analyses suchas flow cytometry. In some applications, gene chips are implemented forgenetic analysis using small sample volumes at the imaging field (i.e.,below the capillary 310 at an implanted specimen). Certain applicationsinvolve RNA/DNA analysis, which may also involve the use of gene chips.In other applications, a flow cytometry approach involving cellularanalysis to classify blood cells is implemented with basic immunologyand virology analysis approaches and is selectively implemented withcellular fluorescence methods. In some implementations involving flowcytometry, green fluorescent protein (GFP)-expressing cells are used toreveal protein concentrations and/or protein-protein interactions usingfluorescence resonance energy transfer (FRET). In various otherapplications, cells or molecules within cells to be analyzed are labeledusing a variety of techniques so that they emit fluorescence; thesetechniques may include, for example, antibody labeling, geneticexpression of a fluorophore-linked protein and others. In addition tothe above, various fluorescent-based analysis approaches are implementedusing sampled fluid with post-sampling fluorescent labeling.

Other applications involve biochemical assays such as those involvingmicrodialysis (a biochemical analysis approach) to analyze fluids forsmall metabolites in conjunction with micro-imaging of a sample via thecapillary 310. In some applications, the microdialysis approachimplements small hypodermic cannulae attached to the capillary 310 foraccess to a sample or specimen in which the lower end 312 is embedded.One microdialysis approach involving the capillary 310 is directed tothe examination of aggregate neurotransmitter levels in a live rodentbrain. Other microdialysis approaches are directed towardscharacterizing ischemia and head injury, and/or cardiac or pepaticdamage or disease.

In one example embodiment, a fluid exchange 345 is implemented with fine(e.g., 31-35 gauge) hypodermic tubing, such as stainless steel tubing,that runs along the side of the capillary 310, for insertion into asample. This fluid exchange may, for example, be implemented with, orinstead of, the fluid exchange 340. In some applications, the fluidexchange 345 is integrated with the capillary 310, as shown by dashedlines 347.

In another example embodiment, the arrangement 300 includes anelectrical exchange 350 that is adapted to provide an electricalstimulus to a sample and/or detect an electrical characteristic of asample in which the lower end 312 of the arrangement 300 is embedded. Insome applications, the arrangement 300 includes one or more electrodesnear the lower end 312 (e.g., near a portion of the exchange 350adjacent the lower end 312). When an electrical stimulus is to beapplied to the sample, combined electrophysiological and micro-opticalmeasurements are facilitated, with the electrodes implemented tofacilitate the application of the stimulus. Similarly, where anelectrical response is to be detected, the electrodes are implemented todetect such a response.

In some applications, the electrical exchange 350 is implemented todetect an electrical response of a sample in which the arrangement 300is embedded. For example, electrodes 360 and 362 near the lower end 312may be implemented to detect an electrical response to an opticalstimulus applied via the light director 330. This electrical response ispassed to an external detection arrangement, such as a computer or otherprocessing device, which processes the response and generates a signalor other output that is useful for analyzing characteristics of thesample. For example, images or other characteristics of the sample canbe used to analyze the sample or, where used in living samples, to guidesurgical procedures.

Certain applications involving the detection of an electrical responsefrom a sample having electrically excitable tissues, aggregate activityin cell populations is observed via measurement of electrical fieldpotentials at electrodes 360 and 362 near the lower end 312 of thecapillary 310. Electrocardiogram (ECG), electromyogram (EMG), andelectroencephalogram (EEG) analyses are selectively implemented withsuch applications, using electrical characteristics detected from thesample.

In one implementation, the arrangement 300 includes an electrical probehaving electrodes (e.g., tungsten) inserted through a hypodermic tube,fixed to the side or extending through a lower portion of the capillary310, such as with the electrical exchange 350 or with an external tube355. This probe is implemented to apply an electrical stimulus, and/orto facilitate the detection of an electrical characteristic from asample in which the capillary 310 is embedded. In one implementation,the probe includes two or more electrodes in a differential arrangementthat facilitates field recordings of aggregate neural activity viavoltage changes (e.g., in the range of about 10 μV-2 mV), with commonmode fluctuations mutually cancelled.

The arrangement of the electrodes is selected to facilitate theparticular application, select stimulus approaches or select responseapproaches. In one application, the electrodes are insulated with ameasurement end exposed and having a metal tip (e.g., of about 5 μm inradius). The tips of the electrodes are arranged between about 100-500μm below the lower end 312 of the capillary 310 (e.g., via theelectrical exchange 350 or the external tube 355 extending below asshown). In certain applications, the electrode tips are curved inwardstowards the capillary 310, such that the tip resides near to but outsidethe field of view of the light director 330.

A differential preamplifier 370 is selectively positioned near thearrangement 300 and adapted to receive signals from electrodes asdiscussed above. The preamplifier 370 includes a ground wire 372 thatcan be inserted into a sample animal near to a target below thecapillary arrangement 300. Signals from the preamplifier 370 areprovided to an amplifier and, in turn, to a computer for digitization,with electrical and image data acquisition synchronized at the computer.This approach may be implemented using, for example, the processor 132in FIG. 1 or an arrangement as shown in FIG. 4 and discussed furtherbelow.

In some electrical detection applications, electrophysiologicalrecordings are used to study small animal models of disease. Forinstance, an EMG approach provides signatures of muscle contraction,with various applications directed to the examination of disuse atrophyin rats and the examination of mouse models for human cardiacarrhythmias. An EEG approach is implemented for measuring aggregateneuronal activities in the brain, and is selectively used to examinemouse models for sleep deprivation and seizures. Electrical signals areused as a diagnostic for bodily functions that might be fruitfullystudied at the cellular level with fluorescence micro-imaging. In oneparticular application, epilepsy is analyzed using an EEG approach, withaberrant patterns of Ca²⁺ dynamics in epileptic neurons studied in vivo(via the capillary 310) with combined electrical measurements andmicro-imaging.

FIG. 4 shows an arrangement 400 for analyzing samples, according toanother example embodiment of the present invention. The arrangement 400includes a capillary arrangement 440 implemented, for example, in amanner commensurate with that discussed in connection with the capillaryarrangement shown in FIG. 3. The capillary arrangement 440 includes acapillary tube 442 that is adapted for implantation into a sample 405for analyzing a target region 406 in the sample over time, and acceptsan optical probe 430 that can be inserted from outside the sample toanalyze the target region.

The optical probe 430 is coupled to receive light from a light source410 via a beam splitter 420 and light-passing arrangements 412 and 422,and passes the received light to the target region 406 in the sample405, via a lower portion of the capillary tube 442. Where appropriate,the lower portion of the capillary tube 442 includes one or more lensesor other light-passing arrangements as described, for example, inconnection with FIG. 2. Light from the sample 405 is passed via thecapillary tube 442 into the optical probe 430, and via the light-passingarrangement 422 to the beam splitter 420, which directs the sample lightto a light sensing arrangement 480 via a light-passing arrangement 482.The light sensing arrangement receives the light, generates a signal inresponse thereto and passes the signal to a processor 470 for analyzingthe light.

An electrical probe line 444 is coupled to the capillary tube 442 andadapted for insertion into the sample 405. Depending upon the particularapplication, the electrical probe line 444 is selectively implementedwith a hypodermic type of tube with a material such as that describedabove in connection with FIG. 3. The electrical probe line 444 isadapted to receive one or more electrodes, which are inserted into thesample via the probe line and pass an electrical signal to apreamplifier 450, which is further coupled to the sample via a ground452. The preamplifier 450 amplifies a signal representing an electricalcharacteristic of the sample detected via the probe in the probe line444 and passes the amplified signal to an electrical signal processor460, which processes the signal and passes an output to the processor470 for analysis. In certain applications, additional electrical probelines are implemented with the arrangement 400. Furthermore, certainapplications involve the use of the electrical probe line 444 and/oradditional electrical probe lines to provide an electrical stimulus tothe sample 405.

A fluid probe line 446 is adapted to pass fluid between a fluid system490 and the sample 405. In this manner, fluid is provided to and/ordrawn from the sample 405 near the target region 406. In certainapplications additional fluid probe lines are implemented with thearrangement 400. Furthermore, in some applications involving multiplefluid probe lines, one or more fluid lines are dedicated for supply orwithdrawal of fluid.

FIG. 5 shows a bottom view of a capillary arrangement 500, according toanother example embodiment of the present invention. The arrangement 500may, for example, be implemented in connection with the capillaryarrangement 440 in FIG. 4. The arrangement 500 includes a capillary tubewith outer and inner walls 510 and 515. Four hypodermic tubes 520-526are coupled to the capillary tube outer wall 510, and selectivelyimplemented for fluid and/or electrical applications. In otherapplications, one or more of the hypodermic tubes 520-526 is adapted toreceive a fiber optic cable for passing light into a sample (e.g., foroblique illumination).

The approaches discussed above in general and in connection with theexample figures are amenable to a variety of applications and furtherwith selected approaches involving various devices, tools or othertesting-type apparatus. In this regard, the following approaches areselectively implemented with one or more of the above examples, inconnection with various example embodiments.

In one example embodiment, a lens implemented with a capillary-typeanalysis approach includes a correction lens that cancels opticalaberrations. The lens is implemented in a manner similar to that shown,for example, with the optical elements 220 near the lower end 212 of thecapillary 210 shown in FIG. 2. This approach facilitates neardiffraction-limited resolution for in vivo imaging of fine biologicalstructures discernible with visible light, such as neuronal dendriticspines, mitochondria and stereocilia.

In one embodiment directed to a GRIN lens approach, chromatic andwavefront aberrations present in GRIN micro-lenses are characterized andused to identify a corrective approach which is implemented with lensesused in obtaining an image from the sample. The wavelength dependence ofthe refractive index profile is numerically modeled, and initialmeasurements are made to ascertain variation of the on-axis index withwavelength. This approach facilitates modeling of the variation of thefocal length of a GRIN objective lens over the visible spectrum. Customcorrection lenses are then fabricated with the opposite chromaticdispersion tendencies of GRIN lenses, and facilitate the correction ofchromatic and wavefront aberrations. Further, when selectivelyimplemented with achromatic doublet lenses, the correction lensesfacilitate the correction of spherical aberrations and coma to firstorder. Other applications involve triplet lenses. Calculations ofspherical aberrations and coma take into account the aqueous environmentand the working distance of the GRIN objective lens. These correctivelenses are implemented, for example, with the optical elements 220 inFIG. 2 (alone or with another lens arrangement), facilitating correctionwith the optical probe 230 where GRIN lenses are implemented.

In one example embodiment, correction lenses are implemented in adoublet arrangement with a crown glass convex lens of relatively lowrefractive index and dispersion combined with a flint glass concave lensof relatively higher index and dispersion. If f₁, f₂ and V₁, V₂ are therespective lens focal lengths and Abbe numbers (a measure of chromaticdispersion), then the Abbe condition for having the same focal length atthe top and bottom of the spectral range is f₁V₁+f₂V₂=0. This approachcan be facilitated by a doublet that has a plano-convex shape, and whichthus suffers minimally from spherical aberration and coma. The Abbecondition of the doublet arrangement is modified so f₁V₁+f₂V₂ is notzero, balancing the chromatic aberrations from our GRIN micro-lenses. Insome applications, this modification of the Abbe condition isfacilitated via choice of lens materials, rather than alteration of thedoublet's shape, inhibiting spherical aberrations. In some applications,separate correction lenses are implemented for one- and two-photonimaging approaches, since the relevant spectral ranges for the differentapproaches are quite distinct. In one implementation, a relay correctionlens is combined with an objective correction lens to facilitatecorrection.

One example imaging technique that may be used in connection with one ormore example embodiments discussed herein involves laser scanningfluorescence microscopy. In laser scanning fluorescence microscopy,focused laser light is scanned across a sample. Fluorescent probesinside of the sample absorb the laser light and emit fluorescence at adifferent wavelength. The fluorescence light is collected and used tovisualize the sample. In some applications, this approach is implementedto facilitate sectioning; that is, the sample may be visualizedsub-surface and at varying depth profiles, with stimulation and responselight passed via a lens at an embedded end of a capillary. In addition,fluorescent probes may be attached to a structure of interest inside ofa biological sample such as a protein, a drug, a sequence of DNA, an RNAsequence or a selected molecule. Laser scanning fluorescence microscopythen allows for visualization of the distribution of said structure ofinterest.

In some applications, laser scanning fluorescence microscopy is combinedwith a nonlinear optical process such as two-photon absorption,typically referred to as two-photon fluorescence microscopy ortwo-photon laser-scanning fluorescence microscopy. With this two-photonmicroscopy approach, a fluorescent probe inside of the sample (i.e., ata location below the implanted end of a capillary) absorbs two-photonsfrom a laser pulse that uses short pulses or pulses in the femtosecondto picosecond range and emits a fluorescence photon at a lowerwavelength. This approach is generally beneficial in reducingphotobleaching and phototoxicity, relative to other conventionalapproaches, and is generally robust to light-scattering inside of thesample.

Other nonlinear optical processes used in connection with variousexamples visualize surface and sub-surface structures inside of a samplevia laser-scanning of a sample with one or multiple lasers (i.e., asdirected by a MEMS mirror) via a capillary-type device. In one instance,a harmonic generation approach such as second-harmonic generation (SHG)or third harmonic generation (THG) is used to generate a nonlinearresponse that is detected and used to garner information about a sample.In other instances, nonlinear processes such as Raman scattering orRaman spectroscopy, or Coherent Anti-Stokes Raman Scattering (CARS) areused in stimulating and analyzing sample.

In the above and other example embodiments of the present invention,various characteristics of light direction approaches (such as thoseinvolving a MEMS mirror implementation and/or associated opticalarrangements) are directed to specific functions relating to thestimulation of and/or detection of nonlinear optical characteristics ina sample. In this regard, certain characteristics of the aboveapproaches and those shown, for example, in FIGS. 1-4 are selectivelytailored for nonlinear type conditions, as discussed further below andotherwise.

In various example embodiments, a nonlinear optical detection systemsuch as that discussed above further includes implantablestimulation-facilitating devices. One application involves the use of asystem including a light source, MEMS mirror, fluorescence probes (forimplantation with a sample) and a wavelength-dependent light collectionapparatus. The fluorescence probes are configured to interact with lightfrom the source, directed by the MEMS mirror via a lower end of acapillary, and to facilitate a nonlinear response of the sample in whichthe probes are implanted. This response includes light that is passed tothe detector via the capillary and is generally characterized in thatthe wavelength of the light is different than the wavelength of thesource light. Other nonlinear optical detection applications involve theuse of devices separate from or in addition to the fluorescence probes,with such devices similarly facilitating the generation of a detectablenonlinear optical response of the sample. These other applications mayinclude, for example, harmonic generation applications, Raman scatteringapplications or Coherent Anti-Stokes Raman Spectroscopy (CARS)applications, e.g., as discussed above.

For general information regarding optical analysis and for specificinformation regarding approaches to analysis that may be implemented inconnection with one or more example embodiments herein, reference may bemade to the following patent documents: U.S. Pat. Nos. 6,485,413 and6,423,956, and U.S. Patent Application Publication Number US2003/0142934; each of these is fully incorporated herein by reference.For example, the mirrors implemented in one or more of these examplepatent documents may be implemented in connection with various exampleembodiments discussed herein. One or more different types of lightsources are implemented, depending upon the application andavailability. In some applications, more than one light direction and/orcollection arrangement is used to provide additional functionality.

While the present invention has been described with reference to severalparticular example embodiments, those skilled in the art will recognizethat many changes, including those discussed in the preceding paragraph,may be made thereto without departing from the spirit and scope of thepresent invention.

1-3. (canceled)
 4. An optical system for imaging a sample in a specimen,comprising: a capillary configured for implantation in said specimen; anoptical probe disposed in said capillary, wherein said optical probe isin optical communication with said sample in said specimen, wherein saidoptical probe is configured to communicate electrical informationbetween said sample and an external arrangement; and said externalarrangement.
 5. The optical system of claim 4, further including ahousing (1) affixed to said specimen and (2) configured and arranged torepeatedly receive the optical probe and provide stable optical accessto a target region of the sample for the optical probe.
 6. The opticalsystem of claim 5, wherein the housing includes one or more opticalelements configured to pass light between the target region and theoptical probe, and wherein the one or more optical elements includes amicro-mirror configured and arranged to direct light to and from thetarget region.
 7. The optical system of claim 4, wherein the externalarrangement includes an image processor configured and arranged togenerate structural subcellular resolution images from light collectedby the optical probe.